A selective inhibitor of the NLRP3 inflammasome as a potential therapeutic approach for neuroprotection in a transgenic mouse model of Huntington's disease

Abstract

Background: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of the CAG repeat in the huntingtin (HTT) gene. When the number of CAG repeats exceeds 36, the translated expanded polyglutamine-containing HTT protein (mutant HTT [mHTT]) interferes with the normal functions of many cellular proteins and subsequently jeopardizes important cellular machineries in major types of brain cells, including neurons, astrocytes, and microglia. The NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome, which comprises NLRP3, ASC, and caspase-1, is involved in the activation of IL-1β and IL-18 and has been implicated in various biological functions. Although the existence of the NLRP3 inflammasome in the brain has been documented, the roles of the NLRP3 inflammasome in HD remain largely uncharacterized. MCC950 is a highly selective and potent small-molecule inhibitor of NLRP3 that has been used for the treatment of several diseases such as Alzheimer's disease. However, whether MCC950 is also beneficial in HD remains unknown. Therefore, we hypothesized that MCC950 exerts beneficial effects in a transgenic mouse model of HD.

Methods: To evaluate the effects of MCC950 in HD, we used the R6/2 (B6CBA-Tg[HDexon1]62Gpb/1J) transgenic mouse model of HD, which expresses exon 1 of the human HTT gene carrying 120 ± 5 CAG repeats. Male transgenic R6/2 mice were treated daily with MCC950 (10 mg/kg of body weight; oral administration) or water for 5 weeks from the age of 7 weeks. We examined neuronal density, neuroinflammation, and mHTT aggregation in the striatum of R6/2 mice vs. their wild-type littermates. We also evaluated the motor function, body weight, and lifespan of R6/2 mice.

Results: Systematic administration of MCC950 to R6/2 mice suppressed the NLRP3 inflammasome, decreased IL-1β and reactive oxygen species production, and reduced neuronal toxicity, as assessed based on increased neuronal density and upregulation of the NeuN and PSD-95 proteins. Most importantly, oral administration of MCC950 increased neuronal survival, reduced neuroinflammation, extended lifespan, and improved motor dysfunction in R6/2 mice.

Conclusions: Collectively, our findings indicate that MCC950 exerts beneficial effects in a transgenic mouse model of HD and has therapeutic potential for treatment of this devastating neurodegenerative disease.

Keywords: Huntington’s disease (HD); Interleukin‐1β (IL‐1β); Mutated huntingtin (mHTT); Nucleotide oligomerization domain-like receptor protein 3 inflammasome (NLRP3 inflammasome).

Fig. 1

MCC950 markedly reduces cytotoxicity in striatal progenitor cells and BV2 microglial cells. A, B BV2 microglia were incubated for 4 h with LPS (1 μg/mL) followed by incubation with MCC950 (1 μM) for 2 h. The cells were then incubated with ATP (1 mM, 24 h). Total lysates of BV2 microglial cells were assessed by Western blot analysis to determine the levels of the NLRP3 and actin proteins. The molecular mass is indicated in kilodaltons. C, D BV2 microglia were incubated for 4 h with LPS (1 μg/mL) followed by incubation with MCC950 (1 μM) for 2 h. The cells were then incubated with ATP (1 mM for 24 h). Cell survival (C) and IL-1β expression levels (D) were measured using the CCK-8 assay and ELISA, respectively. The values of the indicated cells were normalized to those of untreated BV2 cells. *P < 0.05 compared to LPS/ATP treated cells (n = 3). E, F STHdh^Q109 cells were incubated for 24 h with MCC950 (1 μM). Total lysates of STHdh^Q7 and STHdh^Q109 cells were assessed using Western blot analysis. G STHdh^Q7 and STHdh^Q109 cells were incubated for 24 h with MCC950 (1 μM). Cell death was quantified using the CCK-8 assay; the values of the indicated cells were normalized to those of untreated STHdh^Q7 cells. The data are presented as the mean ± SEM from three independent experiments. *P < 0.05, STHdh^Q7 vs. STHdh^Q109 cells; ^#P < 0.05 vs. untreated STHdh^Q109 cells. H BV2 cells were incubated with LPS (1 µg/mL for 4 h) with or without MCC950 for 2 h before stimulation with ATP (1 mM for 24 h). The BV2 medium was then collected and used to culture the STHdh^Q109 cells for an additional 24 h. STHdh^Q7 and STHdh^Q109 cell viability was determined by CCK-8 assay. The data are presented as the mean ± SEM from three independent experiments. *P < 0.05, STHdh^Q7 vs. STHdh^Q109 cells; ^#P < 0.05 vs. untreated STHdh^Q109 cells. I BV2 cells were treated with or without MCC950 (1 μM) and 3-NP (5, 10, and 20 mM) for 24 h. The cell viability was determined using the CCK-8 assay. Data are presented as the mean ± SEM from three independent experiments. *P < 0.05 compared with controls (n = 3)

Fig. 2

MCC950 triggers NLRP3 inflammasome assembly in a transgenic mouse model (R6/2) of HD. Mice were treated daily with MCC950 (10 mg/kg of body weight; oral administration) or water for 5 weeks from the age of 7 weeks. The number of NLRP3-positive cells (identified by the expression of NLRP3; green, A) in the indicated mice (water-treated WT mice [n = 6], water-treated R6/2 mice [n = 6], MCC950-treated WT mice [n = 6], and MCC950-treated R6/2 mice [n = 6]) was quantified. Nuclei were stained with DAPI (blue). The histograms show the number of NLRP3-positive cells in the striatum (B). At least 500 cells from each animal were counted. The levels of IL-1β in the serum (C) and striatum (D) were measured by ELISA (n = 3–6 for each condition). The data are presented as the mean ± SEM. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. Scale bars, 20 μm. E–J Striatal lysates were analyzed using Western blotting. The results were normalized to those of actin. Data are presented as the mean ± SEM from three independent experiments. The molecular mass is indicated in kilodaltons. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice

Fig. 3

MCC950 markedly reduces disease progression in a transgenic mouse model (R6/2) of HD. Mice were treated daily with MCC950 (10 mg/kg of body weight; oral administration) or water for 5 weeks from the age of 7 weeks. Rotarod performance (A), clasping (B), and body weight (C) (n = 16–20 for each condition) were assessed. Data are presented as the mean ± SEM. *P < 0.05, WT vs. R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. D Survival assessment (n = 16–20 for each condition; P < 0.05, Kaplan–Meier survival analysis)

Fig. 4

MCC950 significantly reduces neuronal loss and mHTT aggregation in a transgenic mouse model (R6/2) of HD. Mice were treated daily with MCC950 (10 mg/kg of body weight; oral administration) or water for 5 weeks from the age of 7 weeks. A Brain sections of 12-week-old mice were stained for NeuN and EM48. The number of neurons (as identified by the expression of NeuN; green) and the level of mHTT aggregation (EM48; red) in the striatum of the indicated mice (water-treated WT mice [n = 6], water-treated R6/2 mice [n = 6], MCC950-treated WT mice [n = 6], and MCC950-treated R6/2 mice [n = 6]) were quantified. Nuclei were stained with DAPI (blue). The histograms show the number of striatal neurons (B) and the integrated intensity of mHTT (C). At least 500 cells from each animal were counted. Data are presented as the mean ± SEM. Scale bars, 20 μm. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. D–G Striatal lysates were analyzed using Western blot analysis. The molecular mass is indicated in kilodaltons. Results were normalized to those of actin. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. H Striatal lysates were analyzed using a glutathione assay. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice

Fig. 5

MCC950 inhibits microglial activation in a transgenic mouse model (R6/2) of HD. Mice were treated daily with MCC950 (10 mg/kg body weight; oral administration) or water for 5 weeks from the age of 7 weeks. A Brain sections of 12-week-old mice were stained against Iba-1. The number of microglia (identified by the expression of Iba-1; green) in the striatum of the indicated mice (water-treated WT mice [n = 6], water-treated R6/2 mice [n = 6], MCC950-treated WT mice [n = 6], and MCC950-treated R6/2 mice [n = 6]) were quantified. Nuclei were stained with DAPI (blue). The histograms show the integrated intensity of striatal microglia (B). At least 500 cells from each animal were counted. Data are presented as the mean ± SEM. Scale bars, 20 μm. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. C, D Striatal lysates were analyzed using Western blot analysis. The molecular mass is indicated in kilodaltons. Results were normalized to those of actin. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice

Fig. 6

MCC950 inhibits astrocyte activation in a transgenic mouse model (R6/2) of HD. Mice were treated daily with MCC950 (10 mg/kg body weight; oral administration) or water for 5 weeks from the age of 7 weeks. A Brain sections of 12-week-old mice were stained against GFAP. The number of astrocytes (identified by the expression of GFAP; red) in the striatum of the indicated mice (water-treated WT mice [n = 6], water-treated R6/2 mice [n = 6], MCC950-treated WT mice [n = 6], and MCC950-treated R6/2 mice [n = 6]) were quantified. Nuclei were stained with DAPI (blue). The histograms show the integrated intensity of striatal astrocytes (B). At least 500 cells from each animal were counted. Data are presented as the mean ± SEM. Scale bars, 20 μm. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. C Striatal levels of TNF were measured using ELISA (n = 3–6 for each condition). Data are presented as the mean ± SEM. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice. D–E Striatal lysates were analyzed using Western blot analysis. The molecular mass is indicated in kilodaltons. Results were normalized to those of actin. *P < 0.05, between WT and R6/2 mice; ^#P < 0.05 vs. water-treated R6/2 mice

Fig. 7

Schematic representation of signaling pathways that mediate the action of MCC950 in rescuing the detrimental effect of the NLRP3-dependent pathway in the presence of mutant HTT. In the current study, we demonstrated that expression of polyQ-expanded mHTT enhanced the activation of the NLRP3 inflammasome in striatal progenitor cell lines (STHdh^Q7and STHdh^Q109) and a transgenic mouse model of HD (R6/2 mice). Long-term treatment of HD mice with a NLRP3 inhibitor (MCC950) not only reduced the activation of the NLRP3 inflammasome but also rescued neuronal survival and reduced gliosis in a transgenic mouse model (R6/2) of HD. Importantly, oral administration of MCC950 markedly reduced disease progression in R6/2 mice. Collectively, these findings indicate that MCC950 has therapeutic potential for the treatment of HD. Further characterization of the functional role of MCC950 in HD, as proposed herein, will provide important insights that could help facilitate the development of neuroprotective strategies targeting the bioenergetic defects of HD and other aggregate-related diseases